Antisense reporter system for assaying RNA virus replication

ABSTRACT

Reporter systems for assaying positive sense RNA virus replication are provided. The reporter systems comprise a reporter gene in antisense orientation, flanked by the complements of 5′ and 3′ viral genome ends, such that exposure to an RNA-dependent RNA polymerase results in the generation of mRNA encoding an active reporter protein. Such systems may be used, for example, to detect active RNA virus and to monitor RNA virus therapies.

TECHNICAL FIELD

The present invention relates generally to the detection of RNA viruses.The invention is more particularly related to plasmid systems comprisingan antisense reporter construct, which may be used to assay positivesense RNA virus replication.

BACKGROUND OF THE INVENTION

Many pathological conditions affecting humans and other animals arecaused by infection with an RNA virus. Such viruses may be positivesense viruses (in which the viral genome has the same polarity as viralmRNA and may be directly translated), negative sense viruses (in whichthe viral genome is the complement of viral mRNA, and must betranscribed prior to translation) or double-stranded RNA viruses.Positive sense viruses include flaviviruses (e.g., hepatitis C virus);togaviruses (e.g., rubella virus, Sindbis virus, eastern and westernencephalitis viruses) and picornaviruses (e.g., the enterovirus, poliovirus, coxsackievirus, echovirus and rhinovirus).

In order to accurately diagnose and treat conditions caused by positivesense RNA virus infection, assays that are capable of sensitivedetection of such viruses are needed. Most such assays focus ondetecting the presence of viral nucleic acid and/or antigens. Suchsystems have the disadvantage that they cannot distinguish betweenactive virus (capable of replication) and inactive virus, and they oftenlack the sensitivity that is necessary for a clinically reliable assay.

More recently, detection methods that take advantage of unique pathwaysof viral gene expression have been proposed. Such methods rely on theuse of transgenic cell lines in which a virus-specific event triggersthe production of a reporter protein. See Olivo, Clinical MicrobiologyReviews 9:321-334, 1996; Park et al., Proc. Natl. Acad. Sci. USA88:5537-5543, 1991; U.S. Pat. Nos. 5,591,579 and 5,418,132. However, itis unclear whether such systems can detect positive sense RNA viruseswith the necessary sensitivity.

Accordingly, there is a need in the art for a system that permits thesensitive detection of active positive sense RNA viruses. The presentinvention fulfills this need and further provides other relatedadvantages.

SUMMARY OF THE INVENTION

Briefly stated, the present invention provides antisense reportersystems for use in detecting active positive sense RNA viruses. Withincertain aspects, the present invention provides antisense reporterplasmids, comprising a promoter operably linked to a DNA sequenceencoding: (a) a sequence complementary to the 3′ end of a viral genome;(b) a reporter gene in antisense orientation; and (c) a sequencecomplementary to the 5′ end of the viral genome.

Within related aspects, the present invention provides antisensereporter mRNAs encoding: (a) a sequence complementary to a 3′ end of aviral genome; (b) a reporter gene in antisense orientation; and (c) asequence complementary to a 5′ end of the viral genome.

Within certain embodiments of the above antisense reporter plasmids andantisense reporter mRNAs, the viral genome is a Hepatitis C virusgenome. Reporter genes for use within such systems include, but are notlimited to, chloramphenicol acetyl transferase, beta-galactosidase,alkaline phosphatase, green fluorescent protein, human growth factor andluciferase.

The present invention further provides, within other aspects, host cellstransformed or transfected with an antisense reporter plasmid orantisense reporter mRNA as described above.

Within further aspects, the present invention provides methods formonitoring the level of RNA virus replication in an in vitro system,comprising the steps of: (a) contacting a cell as described above with aviral culture; and (b) determining a level of reporter gene expressionin the cell, relative to a predetermined level in the absence of viralculture; and therefrom determining the level of RNA virus replication inan in vitro system.

Methods are also provided, within further aspects, for determining theeffect of an agent on RNA virus replication in an in vitro system,comprising the steps of: (a) contacting a cell as described above with aviral culture and an agent; and (b) determining a level of reporter geneexpression in the cell, relative to a predetermined level in the absenceof agent; and therefrom determining the effect of an agent on RNA virusreplication in an in vitro system.

Within further aspects, methods are provided for determining thepresence or absence of an RNA virus in a sample, comprising the stepsof: (a) contacting a cell as described above with a sample; and (b)determining a level of reporter gene expression in the cell, relative toa predetermined level in the absence of sample, and therefromdetermining the presence or absence of an RNA virus in the sample.Suitable samples include biological samples isolated from a patient.

The present invention further provides, within other aspects, kits fordetermining the presence or absence of an RNA virus in a sample,comprising: (a) an antisense reporter plasmid as described above; and(b) a supply of reagents for detecting expression of the reporter gene.

Within further aspects, the present invention provides methods formonitoring the effectiveness of a therapy for RNA virus infection,comprising: (a) exposing a patient infected with an RNA virus to acandidate therapy; (b) contacting a sample obtained from the patientwith a cell transformed or transfected as described above; and (c)determining a level of expression of the reporter gene, relative to apredetermined level for cells contacted with a second sample obtainedfrom the patient, wherein the second sample was obtained prior to thecandidate therapy, and therefrom monitoring the effectiveness of thecandidate therapy.

Within further aspects, methods are provided for detecting a drugresistant RNA virus, comprising: (a) exposing a sample obtained from apatient infected with an RNA virus to a drug; (b) contacting the samplewith a cell as described above; and (c) determining a level ofexpression of the reporter gene, relative to a predetermined level forcells contacted with a second sample obtained from the patient, whereinthe second sample is not exposed to the drug, and therefrom identifyinga drug resistant RNA virus.

These and other aspects of the present invention will become apparentupon reference to the following detailed description and attacheddrawings. All references disclosed herein are hereby incorporated byreference in their entirety as if each was incorporated individually.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram illustrating the use of a representative antisensereporter plasmid system. A reporter in antisense orientation is insertedbetween hepatitis C virus genome ends, such that a transcript willcontain the complement of the 3′ end of the viral genome, the reporterin antisense orientation and the complement of the 5′ end of the viralgenome. The plasmid is linearized and transcribed in vitro. Theresulting mRNA is transfected into a host cell. In the presence of viraland cellular factors necessary for viral replication and translation,active reporter protein is generated and detected using a suitableassay.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the present invention is generally directed to systemscomprising antisense reporters for use in detecting the presence ofactive positive sense RNA virus. Such plasmids support the transcriptionof mRNA encoding viral genome end complements flanking an antisensereporter sequence. In the presence of active viral RNA-dependent RNApolymerase, the transcript is replicated into an RNA molecule that canbe translated into active reporter protein. Such systems may be used,for example, to detect active RNA virus, as well as to study endogenousfactors involved in viral replication and to screen agents for theability to modulate viral replication. Agents identified using antisensereporter systems provided herein may be used, for example, for thetreatment or prevention of viral infections.

An “antisense reporter plasmid,” as used herein, is a DNA plasmid thatsupports the synthesis of a transcript comprising, in order, (a) acomplement of a 3′ viral genome end; (b) the complement of a reportergene (i.e., the reporter in antisense orientation) and (c) a complementof a 5′ viral genome end. In other words, such a plasmid comprises apromoter operably linked to a sequence that encodes the viral genome endcomplements and antisense reporter gene. Viral genome ends may bederived from any positive sense RNA virus (e.g, Hepatitis C virus,poliovirus, rhinovirus or hepatitis G virus), and may be readilyidentified and cloned by those of ordinary skill in the art. Anantisense reporter plasmid should further comprise an origin ofreplication that permits replication in a desired host cell. Optionally,an antisense reporter plasmid may comprise any of a variety ofadditional components, such as a selectable marker.

Any reporter gene known in the art may be used within such plasmids,provided that expression can be readily monitored. Expression of areporter gene may be determined at the transcriptional level (i.e.,using any of a variety of well known hybridization or amplificationtechniques to assess the level of mRNA transcribed from the reportergene), at the translational level (i.e., using a compound, such as anantibody, that binds to the protein encoded by the reporter gene toassess the level of such protein synthesized in any standard assayformat, such as ELISA) or at the level of activity (i.e., using an assayto measure an activity (e.g., enzymatic) of a protein encoded by thereporter gene). Preferably, reporter gene expression is determined bymeasuring an activity of the encoded protein. Suitable reporter genesinclude, but are not limited to, luciferase (e.g., firefly or Renilla),green fluorescent protein, β-galactosidase and chloramphenicol acetyltransferase, alkaline phosphatase and human growth factor. Antisensereporter plasmids may be maintained in suitable host cells, which may betransformed or transfected using well known techniques.

For use in detecting RNA virus in a sample, antisense reporter plasmidsare generally linearized and transcribed in vitro, to generate antisensereporter mRNA. This reporter mRNA is then transfected into a suitablehost cell. In general, any cell that expresses or can be supplementedwith factors necessary for viral infection may be used as a host cell.Suitable host cells include, but are not limited to, HeLa cells andprimary culture cells. Host cells may be transformed or transfected withan antisense reporter mRNA using standard techniques known in the art tobe appropriate for the particular cell type. Transformed or transfectedhost cells are specifically contemplated by the present invention.

The antisense reporter systems provided herein may be used to determinethe presence or absence of an RNA virus in a sample. Samples that may beassayed include, but are not limited to, biological samples obtainedfrom a warm-blooded animal, such as a human patient. Human biologicalsamples may be, for example, urine, throat secretions, genitalsecretions, breast milk, blood or a fraction of any of the foregoingsamples. A fluid sample may also be prepared from a solid material byextraction or other procedures common in the art. It will be evident tothose of ordinary skill in the art that one or more preparatory steps,such as centrifugation, may be desirable to place a sample in optimalcondition for testing.

To determine whether a sample contains a particular RNA virus, thesample is contacted with a host cell transformed or transfected asdescribed above for a suitable amount of time, and the effect of thesample on reporter expression may be evaluated by measuring the leveland/or activity of the reporter mRNA or protein. Standard techniques maybe employed, such as PCR or hybridization (for evaluating levels ofmRNA) or any of a variety of immunoassays or functional assaysappropriate for the reporter protein employed. In many cases, a reporterprotein is an enzyme capable of detection by routine colorimetric,fluorometric or luminometric techniques, and assays appropriate forparticular reporter genes are well known in the art. For example,luciferase activity may be detected using standard luminometric methods,with luciferin as the enzyme substrate (de-Wet et al., Mol. Cell. Biol.7:725-737, 1987). Commercially available luciferase assays are availablefrom, for example, Promega (Madison, Wis.). Appropriate methods fordetecting the level of expression of other reporter genes will beapparent to those of ordinary skill in the art and may be found, forexample, in Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., 1989 and inmanufacturer protocols.

In general, a signal detected in a reporter protein assay is compared toa signal that corresponds to a predetermined cut-off value. This cut-offvalue is preferably the average mean signal obtained from cellsincubated in the absence of RNA virus. In general, a sample generating asignal that is at least two-fold above the mean is considered positiveis considered positive for RNA virus. The above assay may also be usedto determine the amount of RNA virus in a sample. Absolute levels of RNAvirus may be obtained by comparing the signal generated by a sample withthe signal generated by standards having known amounts of the virus,using techniques well known to those of ordinary skill in the art. Asdiscussed in more detail below, the determination of relative levels ofRNA virus may also be useful in, for example, monitoring a therapy forRNA virus infection or evaluating the response of a RNA virus isolate toa given drug (e.g., in identifying drug resistance).

The antisense reporter systems provided herein may be used to monitorthe level of RNA virus replication in an in vitro system. Within suchmethods, a culture comprising an RNA virus is contacted with a host celltransformed or transfected as described above for a suitable amount oftime, and the level of reporter expression may be evaluated as describedabove. This level is generally determined relative to a predeterminedlevel in the absence of viral culture. This assay can be used forquantitative determination of RNA virus replication or to monitor thechanges in the ability of an RNA virus to replicate over time.

Similarly, an antisense reporter system may be used to determine theeffect of an agent on RNA virus replication in an in vitro system.Within such methods, a culture comprising an RNA virus is contacted withan agent prior to or during contact with a host cell transformed ortransfected as described above for a suitable amount of time. The levelof reporter expression may be evaluated as described above, relative tothe level observed in the absence of agent. The agent may be anycompound whose effect on RNA virus replication is to be determined.Assays for RNA virus replication may be used as primary screens (e.g.,to screen a cDNA expression library or a small molecule combinatoriallibrary) or as secondary screens (i.e., to characterize a particularagent previously identified as a possible antiviral agent).

Detection of RNA virus by the method described above may also be usefulfor identifying drug-resistant RNA viruses and for monitoring therapy.In these aspects, the change in the level of RNA virus in response toexposure to a drug or other therapy is evaluated. To evaluate whether anRNA virus is resistant to a given drug, a sample containing the virus isexposed to a suitable amount of the drug, using methods appropriate forthe sample type which will be apparent to those of ordinary skill in theart. Following exposure, the sample is then tested for the presence ofRNA virus as described above. If the RNA virus in the treated sample isresistant to the particular drug, RNA virus will be detected. It shouldbe noted that the level of drug resistant RNA virus that is detected inthe sample may temporarily decrease in response to treatment.Nonetheless, the RNA virus is considered drug resistant if either thesignal is unchanged after 2-4 weeks of treatment, or if the signalinitially decreases after 2-3 weeks of treatment and thereafterincreases at a follow-up evaluation.

To evaluate the effectiveness of a therapy for RNA virus infection,suitable samples obtained from one or more infected patients are firstevaluated for RNA virus as described above. The candidate therapy isthen applied to the patient(s) and the level of RNA virus followingtreatment is determined. For example, blood drawn from an infectedpatient may be tested for the presence of RNA virus prior to treatment.After 2-4 weeks of treatment, a second blood sample may be drawn andtested for the RNA virus. A therapy is considered effective if thetherapy lowers the level of RNA virus by at least two-fold. Once atherapy is found to be effective, further treatment of patients may bemonitored by performing similar RNA virus assays at intervals of about2-4 weeks until RNA virus is no longer detectable.

Antisense reporter systems as described herein have the advantage overother assays, in that only samples containing active virus (i.e., virushaving an active RNA-dependent RNA polymerase) are detected. Thisresults in a sensitive assay for infectious RNA virus. This assayfurther is suitable for high-throughput screening formats. As schematicdiagram illustrating a the use of an antisense reporter plasmid systemas described herein is provided in FIG. 1.

The present invention further provides kits for use in detecting thepresence of RNA virus in a sample. Such kits typically comprise two ormore components necessary for performing such an assay. Such componentsmay be compounds, reagents and/or containers or equipment. For example,one container within a kit may contain a host cell line suitable fortransfection with an antisense reporter mRNA construct. Alternatively,or in addition, a kit may provide an antisense reporter plasmid, orantisense reporter mRNA, for use within an assay. One or more additionalcontainers may enclose elements, such as reagents or buffers, to be usedin an assay to detect expression of a reporter gene or the level ofplasmid replication.

The following Examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1

Preparation of Antisense Reporter Plasmid

This Example illustrates the preparation of an antisense reporterplasmid system suitable for detecting Hepatitis C virus.

Oligonucleotide primers are designed to amplify in separate reactionsvia PCR the coding region for the firefly luciferase protein, the 5′untranslated region (5′ UTR) of HCV, and the 3′ untranslated region (3′UTR) of HCV. In addition to the sequences required for hybridization andsubsequent amplification of these fragments, the oligonucleotides arealso designed with nonhomologous sequences at their 5′ ends such thatfollowing amplification, digestions by restriction enzymes are performedand ligation of the digested fragments results in the creation of afragment containing sequences in the order 5′ UTR-luciferase-3′ UTR. Asecond PCR reaction is performed to amplify this entire fragment,followed by restriction enzyme digestion to produce ends compatible forcloning into a plasmid vector containing the T7 RNA polymerase promoter(pBluescript SK, available from Stratagene) in an orientation such thatthe 3′ UTR is closest to the site of transcription initiation.Subsequent improvement of this construct can be performed bysite-directed mutagenesis to eliminate the restriction sites introducedbetween the regions, between the T7 promoter and the 3′ UTR, anddownstream of the 5′ UTR.

Example 2

Detection of Hepatitis C Virus

This Example illustrates the use of an antisense reporter plasmid systemfor the detection of Hepatitis C virus.

A plasmid as described in Example 1 is digested by a restriction enzymefor which there is a cleavage site immediately downstream of the 5′ UTRin the reporter plasmid. Following purification of the digested DNA,transcription by T7 RNA polymerase is performed. After the reaction isstopped, DNase is used to digest the DNA template, and the RNA ispurified. The RNA is transfected via a lipid-based method into cellswhich express proteins supporting replication of the parent virus. Cellsare lysed after a suitable period of time (12-48 hours), and luciferaseactivity is determined by standard methods. Only cells that canreplicate the RNA molecule and translate the resultant RNA complementgive a positive luciferase signal.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for the purposeof illustration, various modifications may be made without deviatingfrom the spirit and scope of the invention. Accordingly, the presentinvention is not limited except by the appended claims.

What is claimed is:
 1. An antisense reporter plasmid, comprising apromoter operably linked to a DNA sequence encoding: (a) a sequencecomplementary to the 3′ end of a viral genome; (b) a reporter gene inantisense orientation; and (c) a sequence complementary to the 5′ end ofthe viral genome.
 2. An antisense reporter plasmid according to claim 1,wherein the viral genome is a Hepatitis C virus genome.
 3. An antisensereporter plasmid according to claim 1, wherein the reporter gene encodesa protein selected from the group consisting of chloramphenicol acetyltransferase, beta-galactosidase, alkaline phosphatase, green fluorescentprotein and human growth factor.
 4. An antisense reporter plasmidaccording to claim 1, wherein the reporter gene encodes a luciferase. 5.A host cell transformed or transfected with an antisense reporterplasmid according to claim
 1. 6. A cell according to claim 5, whereinthe cell is present within a primary cell culture.